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G D Heda

Publications and source records attributed to G D Heda.

12 recordsLinked to original sources
PubMed2001

The Delta F508 mutation shortens the biochemical half-life of plasma membrane CFTR in polarized epithelial cells.

Although the biosynthetic arrest of the DeltaF508 mutant of cystic fibrosis transmembrane conductance regulator (CFTR) can be partially reversed by physical and chemical means, recent evidence suggests that the functional stability of the mutant protein after reaching the cell surface is compromised. To understand the molecular basis for this observation, the current study directly measured the half-life of Delta F508 and wild-type CFTR at the cell surface of transfected LLC-PK(1) cells. Plasma membrane CFTR expression over time was characterized biochemically and functionally in these polarized epithelial cells. Surface biotinylation, streptavidin extraction, and quantitative immunoblot analysis determined the biochemical half-life of plasma membrane DeltaF508 CFTR to be approximately 4 h, whereas the plasma membrane half-life of wild-type CFTR exceeded 48 h. This difference in biochemical stability correlated with CFTR-mediated transport function. These findings indicate that the Delta F508 mutation decreases the biochemical stability of CFTR at the cell surface. We conclude that the Delta F508 mutation triggers more rapid internalization of CFTR and/or its preferential sorting to a pathway of rapid degradation.

Animals↗
PubMed2001

The intestine expresses pancreatic triacylglycerol lipase: regulation by dietary lipid.

We identified the enzyme responsible for alkaline lipolysis in mucosa of rat small intestine. RT-PCR was used to amplify a transcript that, by cloning and sequencing, is identical to pancreatic triacylglycerol lipase. In rats fed normal laboratory chow, pancreatic triacylglycerol lipase mRNA was detected in all four quarters of the small intestine, with the first quarter expressing about three times as much of this transcript as was found in the more distal three-quarters combined. Both acutely and chronically administered dietary fat were shown to regulate pancreatic triacylglycerol lipase mRNA expression and lipase activity. The synthesis of pancreatic triacylglycerol lipase protein by the small intestine was demonstrated by in vivo radiolabeling experiments using [(35)S]methionine/cysteine followed by immunoprecipitation with an anti-pancreatic triacylglycerol lipase antibody. Immunohistochemical studies suggest that pancreatic triacylglycerol lipase protein expression is restricted to enterocytes throughout the small intestine. To our knowledge, this is the first report identifying rat small intestinal mucosa as a site of pancreatic triacylglycerol lipase synthesis and the first demonstration of its modulation in the mucosa by dietary fat. We propose that pancreatic triacylglycerol lipase is used by the intestine to hydrolyze the mucosal triacylglycerol that is not transported in chylomicrons.

Amino Acid Sequence↗
PubMed2000

Surface expression of the cystic fibrosis transmembrane conductance regulator mutant DeltaF508 is markedly upregulated by combination treatment with sodium butyrate and low temperature.

The DeltaF508 gene mutation prevents delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) to the plasma membrane. The current study examines the biochemical basis for the upregulation of DeltaF508 CFTR expression by sodium butyrate and low temperature. Surface CFTR protein expression was determined by quantitative immunoblot following surface biotinylation and streptavidin extraction. CF gene expression was measured by Northern analysis and CFTR function by forskolin-stimulated (125)I efflux. Butyrate increased DeltaF508 mRNA levels and protein expression but did not increase the biochemical or functional expression of DeltaF508 CFTR at the cell surface. Low temperature increased the biochemical and functional expression of DeltaF508 CFTR at the cell surface but did not increase CFTR mRNA levels. Combining treatments led to a synergistic increase in both DeltaF508 mRNA and surface protein levels that results from the stabilization of CFTR mRNA and protein by low temperature. These findings indicate that surface expression of DeltaF508 CFTR can be markedly enhanced by carefully selected combination agents.

Animals↗
PubMed1996

Phosphatase 2A participates in interferon-gamma's induced upregulation of C1 inhibitor mRNA expression.

C1 inhibitor (C1 INH) is the major inhibitor of the proteolytically active subcomponents of C1, kallikrein, activated forms of factor XII, and factor XIa in plasma. We determined the mechanism(s) how interferon-gamma (IFN-gamma) regulates C1 INH mRNA expression in HepG2 cells. Cycloheximide or anisomycin treatment alone did not increase C1 INH mRNA nor did it potentiate C1 INH mRNA expression after IFN-gamma stimulation. C1 INH mRNA levels on Northern blot from untreated and IFN-gamma-treated cells did not change for more than 20 hours after actinomycin D treatment. Actinomycin D and 5,6-dichloro-1-beta-ribofuranosylbenzimidazole abolished IFN-gamma-induced C1 INH mRNA expression. Relatively more C1 INH mRNA precursor (heterogeneous nuclear RNA [hnRNA]) was detected in total RNA from IFN-gamma-treated HepG2 cells than unstimulated cells. Treatment of HepG2 cells with the phosphatase 1 and 2A inhibitors, okadaic acid (> or = 50 nmol/L) and calyculin (> or = 25 nmol/L), decreased IFN-gamma's ability to upregulated C1 INH mRNA. The phosphatase 2A inhibitor, cantharidin (> or = 10 micromol/L), also blocked the IFN-gamma induction of the C1 INH gene. In HepG2 cells total phosphatase 2A activity was significantly increased by C6 ceramide but not IFN-gamma. However, C6 ceramide itself did not increase C1 INH mRNA expression. These data indicate that phosphatase 2A is required to dephosphorylate a substrate in order for IFN-gamma to induce the transcriptional upregulation of C1 INH mRNA, but phosphatase 2A is not a direct stimulator of C1 INH gene expression.

Complement C1 Inactivator Proteins↗
PubMed1993

Expression of platelet C1 inhibitor.

Human platelets contain a pool of C1 inhibitor (C1 INH) distinct from that in plasma. Twelve normal platelet samples washed by centrifugation had a mean platelet C1 INH antigen level of 19.3 +/- 2.8 ng (mean +/- SEM) per 10(8) platelets. These values contrast with the mean +/- SEM platelet C1 INH antigen level of 6.1 +/- 0.9 per 10(8) platelets from 12 C1 INH-deficient patients. The level of platelet C1 INH correlated (r = .7) with the level of plasma C1 INH in normal individuals and patients with classic hereditary angioedema. Platelet C1 INH, like plasma C1 INH, was a 105-Kd protein on immunoblots of solubilized platelets and in thrombin- or collagen-induced platelet releasates. On indirect immunofluorescence, morphologically and immunochemically identifiable elutriated human megakaryocytes had C1 INH antigen. Using nested primer polymerase chain reaction, C1 INH mRNA was detected in megakaryocytes. When activated, human platelets expressed a portion of their total pool of C1 INH antigen on their membrane. Using a competitive enzyme-linked immunosorbent assay for C1 INH as a quantitative, indirect antibody consumption assay, the surface of unstimulated platelets had 0.55 +/- 0.4 ng C1 INH/10(8) platelets (mean +/- SEM). When activated with thrombin, platelets secreted 7.37 +/- 2.2 ng C1 INH/10(8) platelets into the suspension buffer and simultaneously expressed 4.4 +/- 1.2 ng C1 INH/10(8) platelets on their external membrane. These studies showed that activated platelets secreted 38% of their C1 INH and externalized another 23% of the total platelet C1 INH on their membrane. Furthermore, in 125I-anti-C1 INH Fab' binding experiments to platelets, about 8 ng of the antibody fragment specifically bound to 10(8) activated platelets. These data suggest that level of platelet C1 INH packaged into platelet alpha-granules is modulated by the amount of protein produced in megakaryocytes. Platelet alpha-granule C1 INH can both be secreted from platelets and expressed on their activated membranes. The cell membrane expression of C1 INH may be important to modulate the activity of the proteases of the complement and contact systems of plasma proteolysis in the microenvironment of the inflammatory response.

Adult↗
PubMed1990

Interferon gamma increases in vitro and in vivo expression of C1 inhibitor.

C1 inhibitor (C1 INH) is the major protease inhibitor of the first components of the classic complement system and of the proteases of the Hageman factor pathways. Since C1 INH may modulate inflammatory reactions associated with complement and contact system activation, we sought to determine if the cytokine gamma interferon (IFN-gamma) could modulate C1 INH production. Initial studies investigated the effect of IFN-gamma on the molecular and protein expression of C1 INH in human erythroleukemia (HEL) cells. HEL cells constitutively expressed the 2.1 kb mRNA for C1 INH. IFN-gamma (50 to 1,000 U/mL), but not interferon alpha or beta, increased twofold the amount of C1 INH mRNA expressed within HEL cells. Similarly, this cytokine increased HEL cell C1 INH synthesis of a 105 Kd protein 10-fold, from 1.9 +/- 0.5 microgram C1 INH antigen per 10(8) cells (mean +/- SEM) to 19 +/- 8 micrograms/10(8) cells in 8 days. C1 INH produced by HEL cells after IFN-gamma stimulation had fully intact kallikrein neutralizing activity. Moreover, conditioned media of IFN-gamma-treated HEL cells accumulated more secreted C1 INH in 8 days (6.7 micrograms/mL/10(8) cells) than untreated cells (0.6 microgram/mL/10(8) cells). Additional studies were done on plasma specimens from 22 patients with metastatic colorectal carcinoma who received IFN-gamma daily for 4 days by intravenous infusion. Before treatment, the mean +/- SEM C1 INH levels in these patients was 438 +/- 16 micrograms/mL. At day 10 from the start of the infusion, the plasma C1 INH in these patients increased to 586 +/- 32 micrograms/mL (P less than .0001). The extent of rise of plasma C1 INH after IFN-gamma treatment was independent of dose from 0.01 to 40 U/m2. After 30 days, the mean plasma C1 INH levels decreased to 502 +/- 27 micrograms/mL. These combined studies indicate that IFN-gamma can increase C1 INH protein expression in vitro and in vivo.

Blotting, Northern↗
PubMed1989

Synthesis and expression of C1 inhibitor by human umbilical vein endothelial cells.

The biologic activity of C1 esterase, activated forms of factor XII and kallikrein at sites of vascular inflammation may be regulated by C1 inhibitor (C1 INH) elaborated by endothelial cells. Therefore, we investigated whether human umbilical vein endothelial cells (HUVEC) in culture produce C1 INH. Passaged HUVEC contain 1.6 +/- 0.8 micrograms of C1 INH/10(8) cells (mean +/- S.D.; n = 7) which was immunochemically similar to plasma C1 INH measured by a competitive enzyme-linked immunosorbent assay. Methylamine-treated lysates of HUVEC contained a functional inhibitor of purified kallikrein (2.7 +/- 0.8 micrograms activity/10(8) cells, mean +/- S.D.; n = 4). The HUVEC-derived kallikrein inhibitory activity was mostly C1 INH because it was reversed by chemically treating the lysate with chloroform and was neutralized by anti-C1 INH antibody. A lysate of HUVEC derived from an umbilical cord from a patient with Type I hereditary angioedema contained less than 30% of the normal levels of C1 INH antigen and activity. Immunohistochemical staining of HUVEC demonstrated a diffuse pattern of staining for C1 INH. HUVEC C1 INH was also expressed on the endothelial cell surface as detected by binding of anti-C1 INH antibody to intact monolayers and was elaborated progressively into the overlying media over the first 24 h in culture. HUVEC incubated with [35S] methionine secreted a metabolically labeled protein having a molecular mass of 92 kDa immunoisolated using polyclonal or monoclonal antibodies to human C1 INH. A mRNA transcript encoding for C1 INH was detected by slot blot hybridization. Incubation of HUVEC with gamma-interferon stimulated the expression of the 2.1 kilobase mRNA for C1 INH and increased the level of C1 INH produced by these cells. Production and expression of C1 INH by endothelial cells may help modulate the complement system and the contact system of plasma proteolysis on the vascular surface in vivo.

Blotting, Northern↗
PubMed1989

Purification and characterization of the thermostable ribulose-1,5-bisphosphate carboxylase/oxygenase from the thermophilic purple bacterium Chromatium tepidum.

The Calvin cycle enzyme ribulose-bisphosphate carboxylase/oxygenase has been purified and characterized from the thermophilic and obligately anaerobic purple sulfur bacterium, Chromatium tepidum. The enzyme is an L8S8 carboxylase with a molecular mass near 550 kDa. No evidence for a second form of the enzyme lacking small subunits was obtained. C. tepidum ribulose-bisphosphate carboxylase/oxygenase was stable to heating to temperatures of 60 degrees C and could be readily purified in an active form at room temperature. Both carboxylase and oxygenase activities of this enzyme were Mg2+-dependent and carboxylase activity was sensitive to the effector 6-phosphogluconic acid. The Km for ribulose bisphosphate for the carboxylase activity of the C. tepidum enzyme was substantially higher than that observed in mesophilic Calvin cycle autotrophs. Amino acid composition and immunological analyses of C. tepidum and Chromatium vinosum ribulose-bisphosphate carboxylases showed the enzymes to be highly related despite significant differences in heat stability. It is hypothesized that thermal stability of C. tepidum ribulose-bisphosphate carboxylase/oxygenase is due to differences in primary structure affecting folding patterns in both the large and small subunits and is clearly not the result of any unique quaternary structure of the thermostable enzyme.

Centrifugation, Density Gradient↗
PubMed1987

Purification and characterization of alpha-amylase from rat pancreatic acinar carcinoma. Comparison with pancreatic alpha-amylase.

alpha-Amylase was purified to apparent homogeneity from normal pancreas and a transplantable pancreatic acinar carcinoma of the rat by affinity chromatography on alpha-glucohydrolase inhibitor (alpha-GHI) bound to aminohexyl-Sepharose 4B. Recovery was 95-100% for both pancreas and tumour alpha-amylases. They were monomeric proteins, with Mr approx. 54000 on SDS/polyacrylamide-gel electrophoresis. Isoelectric focusing of both normal and tumour alpha-amylases resolved each into two major isoenzymes, with pI 8.3 and 8.7. Tumour-derived alpha-amylase contained two additional minor isoenzymes, with pI 7.6 and 6.95 respectively. All four tumour isoenzymes demonstrated amylolytic activity when isoelectric-focused gels were treated with starch and stained with iodine. Two-dimensional electrophoresis, on SDS/10-20%-polyacrylamide-gradient gels after isoelectric focusing, separated each major isoenzyme into doublets of similar Mr values. Pancreatic and tumour-derived alpha-amylases had similar Km and Ki (alpha-GHI) values, but the specific activity of the tumour alpha-amylase was approximately two-thirds that of the normal alpha-amylase. Although amino acid analysis and peptide mapping with the use of CNBr, N-chlorosuccinimide or Staphylococcus aureus V8 proteinase gave comparable profiles for the two alpha-amylases, tryptic-digest fingerprint patterns were different. Antibodies raised against the purified pancreatic alpha-amylase and tumour alpha-amylase respectively showed only one positive band on immunoblotting after gel electrophoresis of crude extracts of rat pancreas and carcinoma, at the same position as that of the purified enzyme. More than 95% of the alpha-amylase activity in the pancreas and in the tumour was absorbed by an excess amount of either antibody, indicating that normal and tumour alpha-amylases are immunologically identical. The presence of additional isoenzymes in the carcinoma, and dissimilarity of tryptic-digest patterns, may reflect an alteration in gene expression or in the post-translational modification of this protein in this heterogeneously differentiated transplantable pancreatic acinar carcinoma.

Amino Acids↗